Pharmacology Biochemisto' & Behavior, Vol. 10, pp. 831-843. Printed in the U.S.A.
Benzodiazepine Receptors: Cellular and Behavioral Characteristics ARNOLD
S. L I P P A , D O N A L D C R I T C H E T T , M A R Y C. S A N O , C L A I R E A . K L E P N E R , GREENBLATT, JOSEPH COUPET AND BERNARD BEER
EUGENE
Medical R e s e a r c h Division, A m e r i c a n C y a n a m i d C o m p a n y , Lederle Laboratories, Middletown Road, 1: River N Y 10965
LIPPA, A. S., D. CRITCHETT, M. C. SANO, C. A. KLEPNER, E. N. GREENBLATT, J. COUPET AND B. BEER. Benzodiazepine receptors: Cellular and behavioral characteristics. PHARMAC. BIOCHEM. BEHAV. 10(5) 831-843, 1979.--Brain specific benzodiazepine receptors appear to mediate the pharmacological properties of benzodiazepines. A neuronal localization for these receptors is suggested by the parallel decrease in the number of benzodiazepine receptors and cerebellar Purkinje cells in "nervous" mutant mice. Electrophysiological results are compatible with an action of benzodiazepines on neuronally localized, physiological receptors. Biochemical, electrophysiologicaland behavioral experiments highlight the possible importance of frontal cortex in mediating the anxiolytic properties of the benzodiazepines. Triazolopyridazines act upon benzodiazepine receptors, increase punished responding and protect against pentylenetetrazole-induced convulsions, but do not produce the side effects associated with benzodiazepines or affect classical neurotransmitter systems. The structural similarities between triazolopyridazines, purines and the indole portion of certain peptides may provide insights into the nature of the endogenous ligand. Cellular characteristics
Behavioral characteristics
AN understanding of the basic mechanisms by which the benzodiazepines exert their anxiolytic effects might provide tools for investigating those neural substrates involved in anxiety. Recently, several investigators have reported the existence of binding sites for 3H-diazepam and 3Hflunitrazepam in several species including humans [6, 8, 13, 29, 30, 31, 40, 41]. These binding sites conform to many of the criteria established for identifying physiological receptors [47]. Brain-specific 3H-diazepam and aH-flunitrazepam binding has a high affinity, is saturable and stereo-specific [6, 8, 13, 29, 30, 31, 40, 41, 46]. Significant correlations have been obtained between the in vivo and in vitro ability of benzodiazepines to inhibit 3H-benzodiazepine binding and the clinical usefulness of these drugs in humans [9, 30, 31, 40, 41]. Significant correlations have also been reported between the ability of benzodiazepines to inhibit '~H-diazepam binding and their ability to inhibit pentylenetetrazole (PTZ)-induced convulsions [9, 30, 41] and to increase punished responding in a conflict situation [14,26], procedures which are highly correlated with the anxiolytic activity of benzodiazepines, F o r these reasons, it has been suggested that these binding sites represent a common receptor mechanism through which benzodiazepines produce their anxiolytic actions. Because of the potential importance of these findings, research in this area has greatly increased. This paper descibes our attempts to delineate some of the cellular and behavioral characteristics of benzodiazepine receptors, CELLULAR LOCALIZATIONOF BENZODIAZEPINERECEPTORS To study the cellular localization of benzodiazepine receptors, we have investigated :~H-diazepam binding in brains
from nervous mutant mice, an autosomal recessive mt which results in a selective degeneration of cerebell~ kinje cells [25]. Purkinje cells are present in normal nu until the mice are approximately 23 days of age, at time the Purkinje cells begin to degenerate, so that days of age, an estimated 90% of the Purkinje cells cerebellar hemispheres and more than 5(1% in the ' have disappeared [22]. At 15-21 days of age, aH-diazepam binding in ceret mutant mice did not differ from that observed in t littermates and approximated adult levels (see Table 60-70 days of age, however, binding was reduced in 1 mice to 17% of normal littermate control values (Ta These results demonstrate an age-related loss ol zodiazepine receptors in the cerebellum of mutant mi~ allel to that observed for the degeneration of cerebell~ kinje cells. Additional experiments in 60-70 day old mice re that the decreased 3H-diazepam binding only occur cerebellum and was due to a decrease in the maximal n of binding sites (Bmax) and not to any change in affinit (see Fig. 1). Since the age-related, regionally specific 3H-diazepam binding sites in nervous mutant mice par the loss of cerebellar Purkinje cells, these results sugge benzodiazepine receptors reside on Purkinje cells. In additional experiments we have also attempted calize benzodiazepine receptors in frontal cortex. Ster ically placed, unilateral coronal knife cuts transections) were made in anesthetized albino rats anterior border of the caudate nucleus. Three week surgery, all animals were decapitated and :~H-diazepan ing in frontal cortex was determined in the operat~
832
LIPPA
TABLE I BENZODIAZEPINE BINDING SITES IN CEREBELLA OF NORMAL AND NERVOUS MUTANT MICE
Age
intact sides. We reasoned that knife cuts would redu diazepam binding if benzodiazepine receptors were 1 either presynaptically on fibers afferent to the frontal or postsynaptically on the cell bodies of fibers efferer the frontal cortex. In fact, 3H-diazepam binding was cantly higher (p
Benzodiazepine Receptors: Cellular and Behavioral Characteristics ARNOLD
S. L I P P A , D O N A L D C R I T C H E T T , M A R Y C. S A N O , C L A I R E A . K L E P N E R , GREENBLATT, JOSEPH COUPET AND BERNARD BEER
EUGENE
Medical R e s e a r c h Division, A m e r i c a n C y a n a m i d C o m p a n y , Lederle Laboratories, Middletown Road, 1: River N Y 10965
LIPPA, A. S., D. CRITCHETT, M. C. SANO, C. A. KLEPNER, E. N. GREENBLATT, J. COUPET AND B. BEER. Benzodiazepine receptors: Cellular and behavioral characteristics. PHARMAC. BIOCHEM. BEHAV. 10(5) 831-843, 1979.--Brain specific benzodiazepine receptors appear to mediate the pharmacological properties of benzodiazepines. A neuronal localization for these receptors is suggested by the parallel decrease in the number of benzodiazepine receptors and cerebellar Purkinje cells in "nervous" mutant mice. Electrophysiological results are compatible with an action of benzodiazepines on neuronally localized, physiological receptors. Biochemical, electrophysiologicaland behavioral experiments highlight the possible importance of frontal cortex in mediating the anxiolytic properties of the benzodiazepines. Triazolopyridazines act upon benzodiazepine receptors, increase punished responding and protect against pentylenetetrazole-induced convulsions, but do not produce the side effects associated with benzodiazepines or affect classical neurotransmitter systems. The structural similarities between triazolopyridazines, purines and the indole portion of certain peptides may provide insights into the nature of the endogenous ligand. Cellular characteristics
Behavioral characteristics
AN understanding of the basic mechanisms by which the benzodiazepines exert their anxiolytic effects might provide tools for investigating those neural substrates involved in anxiety. Recently, several investigators have reported the existence of binding sites for 3H-diazepam and 3Hflunitrazepam in several species including humans [6, 8, 13, 29, 30, 31, 40, 41]. These binding sites conform to many of the criteria established for identifying physiological receptors [47]. Brain-specific 3H-diazepam and aH-flunitrazepam binding has a high affinity, is saturable and stereo-specific [6, 8, 13, 29, 30, 31, 40, 41, 46]. Significant correlations have been obtained between the in vivo and in vitro ability of benzodiazepines to inhibit 3H-benzodiazepine binding and the clinical usefulness of these drugs in humans [9, 30, 31, 40, 41]. Significant correlations have also been reported between the ability of benzodiazepines to inhibit '~H-diazepam binding and their ability to inhibit pentylenetetrazole (PTZ)-induced convulsions [9, 30, 41] and to increase punished responding in a conflict situation [14,26], procedures which are highly correlated with the anxiolytic activity of benzodiazepines, F o r these reasons, it has been suggested that these binding sites represent a common receptor mechanism through which benzodiazepines produce their anxiolytic actions. Because of the potential importance of these findings, research in this area has greatly increased. This paper descibes our attempts to delineate some of the cellular and behavioral characteristics of benzodiazepine receptors, CELLULAR LOCALIZATIONOF BENZODIAZEPINERECEPTORS To study the cellular localization of benzodiazepine receptors, we have investigated :~H-diazepam binding in brains
from nervous mutant mice, an autosomal recessive mt which results in a selective degeneration of cerebell~ kinje cells [25]. Purkinje cells are present in normal nu until the mice are approximately 23 days of age, at time the Purkinje cells begin to degenerate, so that days of age, an estimated 90% of the Purkinje cells cerebellar hemispheres and more than 5(1% in the ' have disappeared [22]. At 15-21 days of age, aH-diazepam binding in ceret mutant mice did not differ from that observed in t littermates and approximated adult levels (see Table 60-70 days of age, however, binding was reduced in 1 mice to 17% of normal littermate control values (Ta These results demonstrate an age-related loss ol zodiazepine receptors in the cerebellum of mutant mi~ allel to that observed for the degeneration of cerebell~ kinje cells. Additional experiments in 60-70 day old mice re that the decreased 3H-diazepam binding only occur cerebellum and was due to a decrease in the maximal n of binding sites (Bmax) and not to any change in affinit (see Fig. 1). Since the age-related, regionally specific 3H-diazepam binding sites in nervous mutant mice par the loss of cerebellar Purkinje cells, these results sugge benzodiazepine receptors reside on Purkinje cells. In additional experiments we have also attempted calize benzodiazepine receptors in frontal cortex. Ster ically placed, unilateral coronal knife cuts transections) were made in anesthetized albino rats anterior border of the caudate nucleus. Three week surgery, all animals were decapitated and :~H-diazepan ing in frontal cortex was determined in the operat~
832
LIPPA
TABLE I BENZODIAZEPINE BINDING SITES IN CEREBELLA OF NORMAL AND NERVOUS MUTANT MICE
Age
intact sides. We reasoned that knife cuts would redu diazepam binding if benzodiazepine receptors were 1 either presynaptically on fibers afferent to the frontal or postsynaptically on the cell bodies of fibers efferer the frontal cortex. In fact, 3H-diazepam binding was cantly higher (p
